Deller Tobias D, Alzubi Jamal, Mosti Laura, Peschers Marie, Gratzke Christian, Wolf Philipp, Cathomen Toni
Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany.
Center for Cell and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany.
Cells. 2025 Aug 28;14(17):1333. doi: 10.3390/cells14171333.
Chimeric Antigen Receptor (CAR) T cell therapy has achieved high response rates in patients with relapsed or refractory hematologic malignancies. However, comparable efficacy in solid tumors remains limited, partly due to poor CAR T cell persistence and immune-mediated rejection. A major contributor, which has hampered the clinical efficacy of CAR T cells in clinical practice, is the immunogenicity of the murine-derived single-chain variable fragments (scFvs) commonly used in CAR constructs. Cell and humoral immune responses to the murine parts of CARs have been implicated in CAR T cell rejection. Here, we describe the generation and in vitro characterization of humanized CAR T cells targeting prostate-specific membrane antigen (PSMA) on prostate cancer cells, based on two distinct murine scFvs (A5 and D7). Humanization improved the germinality index and successfully preserved CAR surface expression. Functional assays demonstrated that humanized PSMA-CAR T cells retained antigen-specific binding, activation and cytotoxicity, differentiation, exhaustion and cytokine secretion profiles comparable to their murine counterparts. These results support the feasibility of humanization as a strategy to reduce immunogenicity without compromising CAR T cell capabilities, providing a foundation for further in vivo validation in solid tumor settings.
嵌合抗原受体（CAR）T细胞疗法在复发或难治性血液系统恶性肿瘤患者中取得了高缓解率。然而，在实体瘤中的疗效仍较为有限，部分原因是CAR T细胞持久性差以及免疫介导的排斥反应。一个主要因素阻碍了CAR T细胞在临床实践中的疗效，即CAR构建体中常用的鼠源单链可变片段（scFv）具有免疫原性。针对CAR鼠源部分的细胞免疫和体液免疫反应与CAR T细胞排斥有关。在此，我们描述了基于两种不同的鼠源scFv（A5和D7），靶向前列腺癌细胞上前列腺特异性膜抗原（PSMA）的人源化CAR T细胞的产生及体外特性。人源化提高了生发指数并成功保留了CAR的表面表达。功能分析表明，人源化PSMA-CAR T细胞保留了抗原特异性结合、激活和细胞毒性，其分化、耗竭和细胞因子分泌谱与鼠源对应物相当。这些结果支持了人源化作为一种在不损害CAR T细胞功能的情况下降低免疫原性的策略的可行性，为在实体瘤环境中进一步进行体内验证奠定了基础。
